Beta-lactamases which hydrolyze the amide bonds of the Beta-lactam ring of sensitive penicillins and cephalosporins are widely distributed among microorganisms, and play an important role in microbial resistance to Beta-lactam antibiotics. Several methods for detecting the presence of microbial Beta-lactamase have been developed. For example, chemical methods for the detection of the enzymatic hydrolysis of the Beta-lactam ring include: (a) the acidimetric method, which employs a pH color indicator to detect the decrease in pH resulting from the formation of a new carboxyl group; (b) the iodometric method, which is based on the decolorization of a starch-iodine complex by the end products of Beta-lactamase hydrolysis, which act as reducing agents to reduce iodine in the complex; and (c) the chromogenic cephalosporin method which is based on a color change following the hydrolysis of a chromogenic cephalosporin substrate (R. B. Sykes and K. Bush, "Physiology, Biochemistry and Inactivation of Beta-lactamases"; Chemistry and Biology of Beta-lactam Antibiotics, Vol. 3: 155-207 1982 Academic Press, New York, R. B. Morin and M. Gorman, Editors). An alternative to the chemical methods is a microbiological assay method which is based on the loss of antibacterial activity following the hydrolysis of the Beta-lactam ring.
Microbial acylases which remove the acyl side chains of susceptible penicillins or cephalosporins are also produced by many microorganisms. The cleavage of acyl side chains from Beta-lactam antibiotics often results in a decrease in pH and reduction of antibiotic activity.
Although microbial Beta-lactamases do not act exclusively on penicillins or on cephalosporins, many show a predominance of penicillinase or cephalosporinase activity. Thus, chemical or microbiological methods which utilize a single Beta-lactam substrate cannot differentiate penicillinase activity from cephalosporinase activity and often give a false negative result for Beta-lactamase activity.
There exists a need for a method which differentiates penicillinase activity from cephalosporinase activity and distinguishes between Beta-lactamase and acylase activity. The present invention fulfills this need and provides other advantages; principally, that of providing an economical alternative to clinical laboratories which test large numbers of microorganisms for Beta-lactamases compared with methods presently available.